Throughout this application various publications are referred to by number in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
Human immunodeficiency virus-1 (HIV) infects mostly immune cells by binding of the viral envelope protein gp120 to the host cellular proteins, CD4 and CCR5 and/or CXCR4, resulting in fusion of the viral envelope with the cellular membrane. To date, in addition to CD4, CCR5 and/or CXCR4, no other membrane cellular proteins have been identified to participate directly in the process of viral entry (1). However, a few studies indicated that upon binding of the virus to its cellular receptors, intracellular Ca2+ levels rise (2-4) and opening of non-selective cation channels as well as calcium activated K+ channels occurs (5), suggesting that signaling and activation of other proteins may be required for infection and replication. Recently, studies in cell lines and peripheral blood mononuclear cells (PBMCs) indicated that ATP release through pannexin1 (Panx1) hemichannels is required for FEW replication (6), supporting the hypothesis that additional host proteins are required for infection/replication.
Hemichannels are plasma membrane channels that can be open at the unapposed cell surface, forming aqueous conduits permeable to ions and small molecules (e.g., ATP, glutamate, NAD+, and PGE2). They allow diffusional exchange between the intra- and extracellular compartments, constituting a route for autocrine/paracrine cellular communication (7). Hemichannels are constituted by the oligomerization of six protein subunits termed connexins (Cxs) or pannexins (Panxs), both highly conserved protein families encoded by 21 or 3 genes in humans, respectively (8, 9). Panx1 hemichannels in concert with purinergic receptors have been described to be important in different immune functions, including cellular activation (10-12), apoptosis (13), stress signals (14), secretion of inflammatory cytokines (15) and HIV replication (6). However, how HIV infection changes the opening of these channels in primary human CD4+ T lymphocytes, one of the main targets of HIV, remains to be elucidated.
Additionally, it is known that macrophages are critical for HIV infection and spread within the host. They are the first cells to become infected and serve as a viral reservoir (45-49). Macrophage infection does not result in cell death and HIV infected macrophages can persist for long periods of time in host tissues even in the presence of combined antiretroviral therapy (50-53). Infected macrophages in the CNS are also important mediators of HIV-associated neurocognitive disorders, secreting inflammatory mediators and neurotoxic proteins that result in CNS dysfunction (54, 55).
HIV infects macrophages by binding of the envelope protein gp120 to CD4 and then CCR5 receptors and subsequent fusion with the host cell membrane (56-58). Binding of gp120 results in increased intracellular calcium and G-protein signaling (59-61) that involve opening of non-selective cation channels and calcium activated potassium channels (62). However, additional specific host proteins that participate in this process need further evaluation.
Purinergic receptors are activated by extracellular ATP and its byproducts, including ADP and UTP, and are classified intro three groups: adenosine receptors (P1), ATP-gated cation channels (P2X), and G-protein coupled receptors (P2Y). Activation of P2X or P2Y receptors causes an increase in intracellular calcium (19). This laboratory's and other results indicate that macrophages predominantly express P2X1, P2X4, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 receptors (64, 65). P2X and P2Y receptors in macrophages are important mediators of the response to host injury, recognizing extracellular ATP as a damage signal that induces inflammation through cytokine release and superoxide formation (66-68). Additionally, recent studies showed that ATP release and purinergic receptor signaling are activated in response to infectious agents (69, 70).
The present invention addresses the need for novel treatments for HIV infection and inhibition of HIV infection and assays for identifying agents therefor.